Biosensor and Method

ABSTRACT

Surface plasmon resonance (SPR) sensor biointerface with a rigid thiol linker layer and/or interaction layer ligand loading with reversible collapse and/or iron oxide nanoparticle sensor response amplification.

CROSS-REFERENCE TO RELATED APPLICATIONS

The following patent applications disclose related subject matter: Ser.No. 09/______, filed ______ (______). These referenced applications havea common assignee with the present application.

BACKGROUND OF THE INVENTION

The invention relates to (bio)chemical sensors, and more particularly tosurface plasmon resonance (SPR) sensors and related methods.

Optical sensors for (bio)chemical detection can provide real-timeanalysis and typically rely on phenomena such as absorption lines,refractive index changes, and specific binding events. A surface plasmonresonance (SPR) sensor measures changes of refractive index in adielectric biointerface on a thin conductor using the dependence of thesurface plasmon wave vector on the refractive index. Thus with abiointerface including specific binding sites for an analyte, theanalyte can be detected quantitatively in a fluid contacting thebiointerface due to the change in refractive index by addition of theanalyte to the biointerface. Green et al, Surface Plasmon ResonanceAnalysis of Dynamic Biological Interactions with Biomaterials, 21Biomaterials 1823-1835 (2000) and Homola et al, Surface PlasmonResonance Sensors: Review, 54 Sensors and Actuators B 3-15 (1999)describe various types for SPR sensors and applications. Useful pairs ofanalyte and analyte-specific ligand include antibody-antigen,lectin-carbohydrate, receptor-ligand, DNA-DNA, et cetera.

FIG. 6 illustrates a total internal reflection SPR sensor whichilluminates a thin (e.g., 50 nm thick) gold sensor film from the insidewith light of wavelength about 800 nm and detects the reflected lightwith a linear photo-diode array. The refractive index of thebiointerface (boundary layer) on the outside of the gold sensor filmdetermines the wave vector of surface plasmon waves at thegold-biointerface: the surface plasmon wave decays exponentially in thedielectric biointerface with a 1/e decay distance of roughly 200 nm. Theinside illumination of the gold film covers a range of incident anglesdue to the geometry of the sensor; and for the angle at which thelight's wave vector component parallel the gold film matches that of asurface plasmon wave, the illumination will be resonantly absorbed toexcite surface plasmon waves. And the linear photo-diode array detectsthe angle at which resonant absorption occurs and, inferentially, therefractive index of the biointerface. Indeed, monitoring the refractiveindex as a function of time during introduction to the biointerface of afluid containing an unknown quantity of analyte allows analysis of thereaction of analyte with the specific binding sites in the biointerface.

Various biointerface structures functionalized with specificanalyte-binding sites have been proposed: self-assembled monolayer (SAM)assembled from thiols with functionalized tail groups, covalentlyimmobilized derivatized carboxymethyl dextran matrix, streptavidinmonolayer immobilized with biotin and functionalized with biotinylatedbiomolecules, functionalized polymer films, and so forth. For example,U.S. Pat. No. 5,242,828 (Biacore) discloses a gold surface with a SAMlinker (barrier) film bound to the gold and with analyte-affinityligands bound (immobilized) to either the SAM directly or to a hydrogelwhich, in turn, is linked to the SAM. The SAM units have the structureX—R—Y where X binds to the gold and may be a sulfide, R is a hydrocarbonchain of length 12-30 carbons (e.g., (CH₂)₁₆) and preferably withoutbranching for close packing, and Y is —OH which binds to derivatizeddextran (the hydrogel). Similarly, Lahiri et al, A Strategy for theGeneration of Surfaces Presenting Ligands for Studies of Binding Basedon an Active Ester as a Common Reactive Intermediate: A Surface PlasmonResonance Study, 71 Analytical Chemistry 777-790 (1999), shows SAMs withunits having structure X—R—Y with X a sulfide, R a hydrocarbon chain,and Y an ethylene glycol chain. And U.S. Pat. No. 6,197,515 discloses aSAM having unit structure X—R-Ch where Ch is a chelating group whichbinds a metal ion that, in turn, binds an analyte-affinity ligand(binding partner).

Linking an extended hydrogel to the linker film increases the bindingcapacity of the surface. For example, derivatized carboxymethyl dextran(molecular weight from 10 to 500 kDa) may be covalently linked to thelinker film as in Löf{dot over (a)}s et al, Methods for Site ControlledCoupling to Carboxymethylated Surfaces in Surface Plasmon ResonanceSensors, 10 Biosensors and Bioelectronics 813 (1995). This hydrogelincreases the binding capacity of the surface by as much as 10-fold. Itrequires charge preconcentration of the ligand into the hydrogel. Thisis done by suspending the ligand to be immobilized in a low ionicstrength buffer at a pH below the isoelectric point of the ligand. Theligand will be positively charged in this buffer and will rapidlyaccumulate within the negatively charged hydrogel. Pre-activation of thehydrogel matrix by activating a fraction of the carboxyl groups resultsin efficient coupling of ligand. The most common activation chemistryemploys a mixture of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) and N-hydroxy-succinimide (NHS). This produces NHSesters that react with amines (indigenous to most pertinacious ligands),and this is most efficient under basic conditions (e.g. pH 8-9).However, many ligands are acidic and will only become positively chargedat very low pH. Therefore, immobilization yields for these ligands arevery low to negligible.

SPR-based biosensors monitor the refractive index that results frombinding of a target analyte at the biointerface. This refractive indexchange is proportional to the molecular mass of the target analyte andthe number of molecules bound. Hence, for small molecules, or lowbinding levels, it is sometimes necessary to amplify this primarybinding response by using a particle labeled secondary reagent. Gu etal, Enhancement of the Sensitivity of Surface Plasmon ResonanceBiosensor with Colloidal Gold Labeling Technique, 5 SupramolecularScience, 695-698 (1998), studies the interaction of Fab′ (human IgGfragment) with a mixture of human IgG and sheep anti-human IgG with SPR;Gu enhances the signal by attaching colloidal gold to the sheepanti-human IgG. The SPR sensor has a biointerface made of a2-mercapto-ethylamine SAM which amide connects to propionate that, inturn, disulfide connects to the Fab′. The colloidal gold particlesincrease the SPR signal by a factor of up to 300 as the sheep anti-humanIgG binds to the Fab′. However, the stability of colloidal gold andother popular colloidal particles is often poor and stabilizers arerequired. Also linkage of molecules to these particles is commonlycomplicated with moderate results.

Cao et al, Preparation of amorphous Fe₂O₃ powder with different particlesizes, 7 J. Mater. Chem. 2447-2451 (1997) describes extension ofSuslick's method of sonication of metal carbonyls to form amorphous ironoxide nanoparticles.

SUMMARY OF THE INVENTION

The present invention provides biointerfaces and SPR sensors plusrelated methods with one or more of the features of a SAM with rigidunits adjacent to the assembly surface, reversible entrapment for ligandloading of an interaction layer, and detection amplification orpreconcentration with amorphous iron oxide nanoparticles.

These have advantages including increased SPR sensor detection easeand/or sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are heuristic for clarity.

FIG. 1 illustrates in cross-sectional elevation view a first preferredembodiment self-assembled monolayer.

FIG. 2 depicts a functionalized SAM interacting with target analyte.

FIG. 3 is a cross-sectional elevation view of a preferred embodimentbiointerface and self-assembled monolayer.

FIGS. 4 a-4 d show a preferred embodiment ligand linkage method.

FIGS. 5 a-5 b illustrate preferred embodiment amplification.

FIG. 6 is a cross-sectional elevation view of an SPR sensor.

FIGS. 7 a-7 e are various preferred embodiment chain types.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Overview

Preferred embodiment surface plasmon resonance (SPR) sensors and methodsinclude preferred embodiment biointerface structures and/or ligandloading methods and/or signal amplification methods. In particular, apreferred embodiment biointerface linker film on a metal surface has thestructure of a self-assembled monolayer (SAM) formed from compounds witha rigid, roughly linear portion adjacent the metal surface. Suitablecompounds are of the structure X—R—Y wherein X is a group such as —S—that binds to gold (or other free electron metal), Y includes functionalgroup(s) for linking (directly or indirectly) ligand(s) which will bindtarget analyte(s), and R provides a rigid carbon chain backbone whichclose-packs upon self assembly. For the linker film of FIG. 1, X is —S—,Y is —CH₂C(CH₂OH)₃, and R is —C₆H₄C═CC₆H₄C═CC₆H₄C═CC₆H₄C—. Theconjugated double and triple bonds of R provide the rigidity, and thepara connections yield a roughly linear structure. Y provides a bulky,hydrophilic end to form a dense surface with active groups for linkingto either ligands or an interaction layer which, in turn, includesligands. The resulting SAM surface will be composed of tightly packedhydroxyl groups forming an ideal linker film. The stability of thesurface chemistry is directly related to the stability of the linkerfilm and is critical to any biointerface applications.

In general, a biointerface may include a hydrogel interaction layeranchored to a SAM linker film or directly to a metal surface and loadedwith immobilized ligands. FIGS. 4 a-4 d illustrate a preferredembodiment reversible entrapment method for loading ligands into such aninteraction layer.

FIGS. 5 a-5 b show preferred embodiment iron oxide nanoparticle colloidfor enhancing SPR response, thereby enhancing the sensitivity of such abiosensor. After binding of available analyte to ligands immobilized inthe interaction layer of a biointerface, the introduction of a colloidalsuspension of such nanoparticles (precoated with a ligand that binds tothe target analyte) will amplify the SPR signal due to the large effectof amorphous iron bound to the biointerface on refractive index. Also,the magnetic properties of the coated ferric oxide nanoparticles permitmagnetic separation prior to

2. Self-Assembled Monolayer (SAM)

FIG. 1 heuristically illustrates in cross-sectional elevation view afirst preferred embodiment self-assembled monolayer (SAM) using thiolswith a rigid portion adjacent the thiol group. First preferredembodiment SPR sensors with gold detection surfaces use such a SAM as alinker film that enables direct attachment of the ligand. The SAM may beassembled from rigid thiols such as HS-(Hydrocarbon Ring)_(n)-Y where nmay be in the range 1-20 and the head group Y has suitable activatablefunctional group(s) or active group(s). For example, the rigid thiol ofFIG. 1 has a backbone including three phenylethynyl units with thegold-attachment end terminated in a thiobenzene and the other endterminated with a head group of tris(hydroxymethyl)ethane. Of course,during self-assembly on the gold surface, the —SH becomes —S—Au.Alternative rigid carbon chains include anthracene-type andphenanthrene-type (fused aromatic rings) backbone, a steroidal-type(fused non-aromatic rings) backbone, or a mixed structure, all with athiol termination. The compound used for self-assembly could be adisulfide such as Y—R—S—S—R—Y instead of a thiol, the —S—S-cleaves forattachment to the gold. Further, alternative X bonds to the gold couldbe —S—S—Au, —Se—Au, . . . , and also more complex sulfur-containinggroups such as —COS—Au, —CSS—Au, . . . .

The resulting SAM's are extremely stable and may be functionalizeddirectly with ligands to form the interaction layer of a biointerface.FIG. 2 heuristically illustrates such an interaction layer with thecrescents representing ligands linked to the linker film SAM and circlesrepresenting analytes. In this case the interaction layer is very thin(only a single monolayer of ligand), but it is possible to extend thislinker film by linking an extended support (e.g. hydrogel) that cananchor a high density of bound ligand to the surface; see FIG. 3.

For the case of ligands directly bound to the SAM, a mixed SAM could beused in which a small fraction (e.g., 5-10%) of the SAM units are longerthan the majority of SAM units, and only these longer SAM units containa head group which binds the ligand. The extra length could be either alonger backbone or a longer head group of a combination. The majority ofSAM units would have an inactive, hydrophilic head group. Thus theligands would be spaced on the SAM and extend beyond the majoritysurface and thereby permit large analytes binding to the ligands withoutsteric hinderence from adjacent ligands. For example, a SAM assembledfrom a mixture of X—R—Y and X—R-Z where X, R, and Y are as in FIG. 1 andZ is —(CH₂)₂₀COOH; or the Y could be hydrophobic such as —CH₃ and the Zhydrophilic and active such as —(CH₂)₁₀(CHOH)₆CH₂OH. More generally, thetwo (or more) compounds for self-assembly could be somewhat different:X—R—Y and X′—R′—Y′ or one or more asymmetrical precursors such asY—R—S—S—R′—Y′.

Interchain interactions of the rigid thiols permits close packing toform the SAM and thus preventing oxidation of the thiol-gold bond andensuring linker film stability. The head group is composed of a clusterof hydroxyl groups which form a hydrophilic surface that resistsnon-specific binding while also allowing activation and furtherderivatization for attachment of ligands (FIG. 2) or hydrogels (FIGS. 3and 4 a-4 d also showing ligands linked to the hydrogel). Other rigidhydrocarbon ring structures (e.g. steroidal compounds, fused ring chainsetc.) may be employed.

Indeed, rigidity of the carbon chain adjacent to the sulfur attachmentat the gold surface underlies linker film stability and may becharacterized as a carbon chain with no sp³-bonded carbons. Also, roughlinearity of the rigid chain helps close-packed assembly. For example,the following classes of rigid carbon chains may be used.

(1) aromatic rings para-connected by linear alkynes, as in FIG. 1.

(2) direct para-connected aromatic rings such as biphenyl; see FIG. 7 c.

(3) fused aromatic rings, such as anthracene, phenanthrene, andchrysene; see FIGS. 7 a-7 b.

(4) fused non-aromatic rings, such as steroid types; see FIG. 7 d.

(5) an ethynyl connecting the sulfur to a ring; see FIG. 7 e.

(6) combinations of (1)-(5).

The rigid carbon chain should have a length in the range of 6-100 carbonatoms, and preferably in the range 10-50. The length is measured as theshortest string of carbons from X to Y, so traversing a para-connectedaromatic ring would count as four carbons.

The first preferred embodiment SAM in FIG. 1 is of class (1) andeffectively has a rigid, roughly linear 22-carbon chain with 16 aromaticcarbons (four in each of the four rings) and 6 sp carbons (two in eachof three —C≡C— connectors). Replacing the —C≡C— groups with trans—CH═CH— groups connecting the rings would somewhat maintain the rigidityand linearity of the carbon chain. The number of carbons in such FIG. 1rigid carbon chains increases in multiples of 6 starting from 10 (twoaromatic rings with one ethynyl between).

Biphenyl provides a chain of length 8 carbons; see FIG. 7 c. Additionalrings increments the length in multiples of 4.

With three fused aromatic rings (anthracene and phenanthrene) the rigidcarbon chain would have 7 or 8 carbons, depending upon the —S- and —Yconnection locations on the terminal rings; see FIGS. 7 a-7 b.

In a FIG. 7 d type chain, rigidity against ring flexing arises from thefusing of multiple rings plus, optionally, double bond(s) in some rings.In particular, for a thio-steroid-based SAM the four fused non-aromaticrings (cyclopentyl hydro-phenanthrene) provide a 9-carbon chain from thesulfur as in FIG. 7 d.

Substitutions such as F or OH in place of H may be made on the rings orthe trans —CH═CH— connectors provided the substitutions do not deterclose packing of the rigid chains. Further, the rigid chains may bepartitioned into two (or more) rigid subchains with a heteroatom or sp³carbon or larger group connecting the two subchains. For example, aphenyl ether derivative —S—C₆H₄—O—C₆H₄—Y has two 4-carbon subchains(each para-connected aromatic ring), and a variant of the rigid chain ofFIG. 1 could have a CH₂ group inserted to form—S—C₆H₄C═CC₆H₄CH₂C≡CC₆H₄C≡CC₆H₄—Y with rigid subchains of lengths 11 and13 (the methylene carbon counts as the terminal carbon in bothsubchains). The preferred minimum length for the rigid carbon (sub)chainconnected to the gold-bonded sulfur (i.e., adjacent the gold) is 8 for asingle chain and 6 for a chain with partitions.

The head group may have functional and/or coupling group(s) connected toa separate (non-rigid) carbon chain such as the trishydroymethly ethylof FIG. 1 or have functional and/or coupling group(s) directly connectedto the terminal of the rigid carbon chain. In particular, Y couldinclude a functional group (useful for attaching an interaction layer ora coupling group) such as hydroxyl, amine, carboxyl, thiol, aldehyde,and mixtures thereof, and/or include coupling group(s) (useful fordirect coupling of ligands to the SAM) such as N-hydroxysuccinimideester, reactive imidazole derivatives, epoxy, aldehyde, sulfonylchlorides, vinyl, divinylsulfone, halogens, maleimide, disulfides,thiols, and mixtures thereof.

For further example, the tris-hydroxymethyl ethyl head group of the FIG.1 has its own 3-carbon methyl ethyl backbones ending with bulky,hydrophilic hydroxyl functional groups. Similar head groups could begem-ethyl diol (—CH₂—CH(OH)₂), fluoro gem-propyl diol (—(CF₂)₂—CH(OH)₂),ethylamide (—CH₂—CONH₂), and other such hydrogen-bonding functionalgroups. Alternatively, the head group could be part of the terminal ofthe rigid carbon chain, such as a terminal phenol or aniline or analogs:one or more —OH's and/or one or more —NH₂'s on the terminal ring. Evenembedding one or more O's or N's in a terminal ring which may be a5-member ring; that is, a terminal such as furan, pyridine, pyrrole,imidazole, quinoline, oxazole, indole, . . . where the ligand bonds to acarbon in the terminal ring or may bond to an embedded N.

In more detail, the first preferred embodiment (FIG. 1) self assembledmonolayer (SAM) using hydrocarbon ring-based thiols can be fabricatedwith the following steps:

-   -   (a) The metal (gold) coated surface of a substrate is cleaned by        exposure to an oxygen plasma and then a hydrogen plasma for 5        min, respectively.    -   (b) A 0.1 mM-10 mM solution of the rigid thiol compound is        prepared in a suitable solvent system (e.g. dichloromethane,        ethanol, hexane etc.), and the cleaned substrates are incubated        in the solution for 48 hours at room temperature. Elevated        incubation temperature from 30 to 100° C. may improve linker        film packing density at the metal surface.    -   (d) The linker film coated surface is rinsed repeatedly in the        solvent used for coating and then in ethanol. The        linker-on-metal coated substrates are then stored under nitrogen        until required.

3. Interaction Layer Loading Method

At its simplest the interaction film will simply be composed of theligand attached directly to the SAM. However, the attachment of anextended hydrogel to support higher loading capacity is favored in manyapplications and is shown in FIG. 3. The hydrogel layer typically is inthe range of 5-200 nm thick. The hydrogel can be composed of anyhydrophilic polymer which provides high biomolecule loading capacityplus low non-specific binding of molecules such as proteins found incrude samples. High ligand loading capacities are achieved by employingthe preferred embodiment ligand loading method which is based on amechanical entrapment principle.

FIGS. 4 a-4 d illustrate the preferred embodiment ligand loading andlinkage mechanism as follows. A hydrogel composed of extended chains(e.g., dextran) is attached to the linker film (SAM) using covalentcoupling as illustrated in FIG. 4 a. The hydrogel is preactivated tointroduce coupling groups (e.g., NHS esters). Then self-associate theseextended chains to form a cross-linked mesh as shown in FIG. 4 b.Ligands (e.g., proteins) exposed (e.g., in solution) to thiscross-linked hydrogel mesh will be entrapped mechanically, resulting inthe accumulation of ligand within the hydrogel where it is covalentlyimmobilized through reaction with the previously-introduced couplinggroups; see FIG. 4 c. Once the ligand is immobilized (coupled), thecross-linking of the hydrogel is reversed resulting in an extendedhydrogel with a high density of immobilized ligand as depicted in FIG. 4d. Residual coupling groups in the hydrogel are blocked. Any techniquesthat reversibly cross-link the hydrogel may be employed. A cleavablecovalent linkage, such as a sulfide bond between adjacent chains issuitable. But other non-covalent interaction may be more suitable forinduction of reversible hydrogel cross-linking.

Example 1

Activation of a hydrogel using N,N′-Carbonyl diimidazole (CDI) causescross-linking of hydroxyls that in close proximity via an activeimidazole carbonate. The activated surface is expose to a proteinsolution (0.1 mg/ml in phosphate buffer, pH 8.0) resulting in entrapmentof protein molecules in the cross-linked hydrogel mesh. The activecarbonate linkage reacts with the primary amines of the proteinresulting in covalent coupling of the protein. Any remaining imidazolecarbonates are inactivated using 1 M ethanolamine, pH 8.5. It isimportant to limit the contact time of the protein with the activesurface to prevent excessive linkages with the hydrogel from forming.

Example 2

The substitution of the amino acid histidine into a dextran hydrogelalready having coupling groups introduces both amidazole and carboxylgroups. Then exposing the hydrogel to a buffer of pH<6.0 will result inthe presence of both positive and negative charges that pair up andprovide a cross-linking of the matrix. Protein in solution is thenentrapped mechanically in the pores formed due to cross-linking andimmobilized via the coupling groups already present. Then increasing thepH will undo this electrostatic cross-linking. More precisely, across-linked state is attained at pH<6.2 and a non-cross-linked state atpH>6.2.

Example 3

The coupling groups may be carboxymethyl and the reversiblecross-linking employs phenyl bis(boronic acid). This reagent interactswith adjacent hydroxyls on a hydrogel for pH in the range 8 to 9,thereby cross-linking any hydrogel possessing a cis-diol (e.g. dextran).After ligand entrapment and immobilization, the cross-linkinginteraction is reversed at pH in the range 3 to 4.

Example 4

Cross-linking employs an affinity interaction such as metal chelatinglinkages where a metal ion receptor (e.g., imidodiacetic acid) and apoly-histidine tag are each linked to the hydrogel matrix. Then thepresence of the appropriate metal ion (e.g., Ni²⁺) will complex with thereceptor and tag to form a crosslink. Whereas, introduction of acompetitive metal chelating agent such as ethylenediamine tetraaceticacid or absence of the appropriate metal ion or changing the pH of thelocal environment reverses the complex formation, and hence, reverseshydrogel cross-linking.

Other coupling groups include reactive imidazole derivatives, epoxy,aldehyde, solfonyl chlorides, divinylsulfone, halogens, maleimide,dusulfides, and thiols. Other cross-linking groups include diols whichare cross-linked by complex formation when exposed to moleculespossessing two or more boronic acid residues. Other cross linkingreversing methods include changing the ionic strength of pH or adding across-linking inhibitor.

4. Amorphous Iron Oxide Nanoparticle Amplification

The signal from an SPR sensor indicating target analyte bound to theimmobilized ligands in the interaction layer can be amplified by afurther step of introducing a second solution or colloidal suspensioncontaining a second ligand which also binds specifically to the analyteand thereby increase the mass bound in the biointerface. That is, ratherthan just measure the refractive index change due to added analyte,measure the refractive index change due to added second ligand plusanalyte. This is analogous to forming a sandwich ofligand1/analyte/ligand2 when analyte is present, and thus increases thesensitivity of the SPR sensor.

A preferred embodiment amplification method uses a second ligand linkedto a nanoparticle of amorphous iron oxide (Fe₂O₃); this not onlyincreases the bound mass but adds highly polarizable material to greatlychange refractive index. For example, the target analyte could be anantigen, and the biointerface could include an immobilized firstantibody to the antigen. Thus when a solution containing an unknownamount of antigen is introduced to the biointerface, any antigen willbind to the first antibody and thereby shift the resonance of the SPRsensor corresponding to the amount of bound antigen. Then, introduce tothe biointerface a suspension of amorphous iron oxide nanoparticles(average diameter 5-100 nm) which have bound secondary antibodies to theantigen. The secondary antibodies (and thus the iron oxidenanoparticles) will bind to any (already-bound) antigen and therebyamplify the resonance shift of the SPR sensor. The resonance shift dueto amorphous iron oxide nanoparticle plus second antibody plus antigenwill be on the order of 100-1000 times the resonance shift due to thetarget analyte alone. Note that an antibody molecule may have size onthe order of 10 nm, so many antibody molecules may be attached to asingle iron oxide nanoparticle, especially when the constant part of theantibody links to the iron oxide to leave the variable partantigen-binding sites open. FIG. 5 a heuristically illustrates thisexample of a sandwich of biointerface-antibody/antigen/antibody-ironoxide nanoparticle.

Further preferred embodiments protect the iron oxide nanoparticles witha coating of (short chain) carboxymethylated dextran which acts as alinker layer for the antibodies (or other analyte binding ligands). Thecarboxyl groups covalently attach to iron oxide, and the coating ofdextran renders the surface stable in aqueous environments and resistantto nonspecific binding. FIG. 5 b illustrates the coated nanoparticleswith linked affinity ligands. The nanoparticles have a low density andsediment very slowly, and when coated with dextran the nanoparticlesform a stable colloid in solution. Indeed, the nanoparticles typicallyare spongelike and porous and appear to be agglomerations of smallerparticles on the order of 10 nm size. Other functional groups forcovalently attaching to the nanoparticles may used in place ofcarboxylic; namely, thiol, hydroxyl, etc. And other materials may besubstituted for the dextran to coat the nanoparticles; for example,other hydrogels that have minimal non-specific binding.

Because the iron oxide nanoparticles exhibit superparamagnetism, thenanoparticles can also be used to magnetically concentrate the antigen(analyte) prior to introduction to the SPR biointerface for measurement.That is, inject affinity-ligand coated amorphous iron oxide nanopartlcesinto a sample containing an unknown amount of analyte; next, pass themixture through a magnetic field to extract the nanoparticles; and thenintroduce the extracted nanoparticles to the SPR sensor biointerface.

In more detail, a preferred embodiment amorphous iron oxide nanoparticlesuitable for amplification may be prepared with the following steps:

-   -   (a) The amorphous iron oxide nanoparticles can be formed by        sonication of Fe(CO)₅ at 20 KHz for 4 hours in a decalin (or        other solvent) solution under an air atmosphere at 0° C. The        nanoparticle size can be increased by increasing the carbonyl        concentration.    -   (b) The particle may be coated with the ligand of interest by        simply suspending the particles in 10 mM phosphate buffer, pH        7.4, containing 0.14 M NaCl and adding solubilized ligand in the        required molar excess. A molar excess of 10:1 ligand to particle        is acceptable. The mixture is incubated at room temperature for        1 h and bovine serum album is then added to a final        concentration of 1 mg/ml to block excess binding sites. Coated        particles may be stored in this solution, containing 0.05%        sodium azide as preservative, at 4° C.    -   (c) Alternatively, the particles may be pre-coated in a short        dextran to improve performance and then link the ligand of        interest. Suspend the particles in 10 mM phosphate buffer, pH        7.4, containing 0.14 M NaCl and add dextran at 1 mg/ml. The        mixture is incubated at room temperature for 1 h. The coated        particles may be recovered by centrifugation and resuspended in        10 mM phosphate buffer, pH 7.4, containing 0.14 M NaCl and 0.05%        sodium azide as preservative. Ligand may be linked to the        dextran coated particles using the appropriate linkage        chemistry.

5. Amorphous Iron Oxide Nanoparticle Applications

The preferred embodiment amorphous iron oxide nanoparticles may also beused for enhancement of various assays. In particular, disposable teststrip (lateral flow) devices where affinity recognition is visualized bythe accumulation of colloidal particles in a localized area giving riseto a visible color intensity change at the area. In addition, thesesuper paramagnetic preferred embodiment nanoparticles are ideal for alltechnologies employing super paramagnetic particles for magneticseparation of target analytes.

6. Systems

Preferred embodiment SPR sensors have the overall structure asillustrated in FIG. 6 plus use a preferred embodiment biointerface as inthe foregoing. Further, preferred embodiment ligand loading methods canbe applied to either preferred embodiment SPR sensors or other SPRsensors with hydrogel interaction layers. Lastly, preferred embodimentiron oxide nanoparticle amplification or enhancement methods can be usedin operation of either preferred embodiment SPR sensors or other SPRsensors with immobilized ligands or other assay systems usingconcentrations, visible or magnetic.

7. Modifications

The preferred embodiments may be modified in various ways whileretaining one or more of the features of a biointerface with a linkerfilm of rigid chains adjacent the surface and a method of interactionlayer ligand loading and amorphous iron oxide amplification/separation.

For example, the SAM structure X—R—Y could be varied so that X couldderive from any of thiol, disulfide, sulfide, selenide, diselenide,thiocarboxyl, and other such groups with a sulfur/selenium for bindingto gold or other free electron metal; and X could include one carbonatom (such as a thiocarboxyl) connected to R; R could be any carbonchain with a rigid structure adjacent the metal and preferably linear;and Y could terminate in any convenient functional group(s), especiallygroup(s) with a cross-sectional diameter comparable to the site spacingon the metal surface to provide a dense SAM surface.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.A method of immobilizing affinity-ligands to a hydrogel, comprising: (a)providing a hydrogel with coupling groups for affinity-ligands and withcross-linking groups; (b) cross-linking said hydrogel using saidcross-linking groups; (c) introducing affinity-ligands to saidcross-linked hydrogel from step (b); and (d) reversing saidcross-linking of step (b) after step (c).
 12. A method according toclaim 11, wherein said coupling group and cross-linking groups areactive carbonates, formed by activation of the hydrogel withN,N′-carbonyl diimidazole, or other carbonylating reagent.
 13. Themethod of claim 11, wherein said coupling group is selected from thegroup consisting of N-hydroxysuccinimide esters, reactive imidazolederivatives, epoxy, aldehyde, solfonyl chlorides, divinylsulfone,halogens, maleimide, dusulfides, thiols, and mixtures thereof.
 14. Themethod of claim 11, wherein the cross-linking groups are diols and arecross-linked by complex formation when exposed to molecules possessingtwo or more boronic acid residues.
 15. The method of claim 11, whereinthe cross-linkable groups are positively and negatively charged and formion pairs that result in cross-linking of the matrix.
 16. The method ofclaim 15, wherein said positively and negatively charged groups areintroduced by linking of histidine to the hydrogel and where across-linked state is attained at pH<6.2 and a non-cross-linked state atpH>6.2.
 17. The method of claim 11, wherein reversible cross-linking ismade to occur through affinity interactions between two or moremolecules linked to the hydrogel, where these cross-linkages are metalchelating linkages wherein both the metal ion receptor andpoly-histidine tag are linked to the matrix and where the presence ofthe appropriate metal ion causes complex formation, and hence,cross-linking of the hydrogel.
 18. The method of claim 11, whereinreversible cross-linking is made to occur through affinity interactionsbetween one or more molecules linked to the hydrogel, where thesecross-linkages are imidodiacetic acid and a poly-histidine tag that areboth linked to the matrix and are complexed by exposure to Ni²⁺ andwhere cross-linking is reversed by addition of any competitive metalchelating agent such as ethylenediamine tetraacetic acid or changing thepH of the local environment.
 19. The method of claim 11, wherein theligand to be immobilized is mechanically entrapped by exposure to theactivated cross-linked hydrogel and where said trapped ligand reactswith the coupling groups of the hydrogel resulting in linkage of theligand to the hydrogel.
 20. The method of claim 11, whereincross-linking of the hydrogel is reversed after linkage of the ligand bychanging the pH of the environment.
 21. The method of claim 11, whereincross-linking of the hydrogel is reversed after linkage of the ligand bychanging the ionic strength of the solution in contact with thehydrogel.
 22. The method of claim 11, wherein cross-linking of thehydrogel is reversed after linkage of the ligand by adding an inhibitor.